1. Field of the Invention
This invention relates to a method and apparatus for imaging and irradiating scattering media.
2. Description of the Related Art
(Note: This application references a number of different publications as indicated throughout the specification by one or more reference numbers in superscript X. A list of these different publications ordered according to these reference numbers can be found below in the section entitled “References.” Each of these publications is incorporated by reference herein.)
Realizing high-resolution fluorescence imaging within scattering biological tissues is a central goal in biomedical imaging. Considerable efforts have been made to extend the imaging depth of optical methods1-7, but focal excitation of fluorescence has so far been fundamentally limited to a depth of one transport mean free path, or approximately one millimeter in most biological samples. This is because conventional focusing approaches treat scattered light as noise and select for the ballistic light component, which exponentially decreases with depth. However, scattered light contains important information about the sample, which can in fact be utilized. When light passes through scattering samples, its wavefront is seemingly randomized, but the randomization occurs in a deterministic and time-symmetric way. These properties of elastic light scattering have recently been used to focus light through turbid samples by iterative wavefront optimization8-15 and by time-reversal using optical phase conjugation10,16-18. These methods are, in many ways, analogous to adaptive optics methods used in astronomy to cancel out the effect of atmospheric scattering19,20. However, in contrast to astronomy where it is sufficient to image through a turbid medium (the atmosphere), the goal of biomedical imaging is to image inside.
To achieve focusing inside tissues, Xu et. al.21 proposed a scheme termed time-reversal of ultrasound encoded light (TRUE), which combines optical phase conjugation22 with ultrasound encoding23. They used focused ultrasound, which is much less scattered than light in biological tissues, to create a virtual source of light frequency-shifted by the acousto-optic effect. Scattered light emanating from this source was then time-reversed by a photorefractive crystal acting as a phase conjugate mirror. Xu et. al.21 inferred the formation of a time-reversed optical focus from a line-scan across millimeter-scale absorbers embedded in tissue-mimicking phantoms.
While promising improved absorption contrast21,24,25, the use of this technique for high-resolution fluorescence imaging in biological tissues remains fundamentally challenging. Because of the low ultrasound modulation efficiency26, the phase conjugate mirror has to provide orders of magnitude higher than unity gain to excite detectable fluorescence. This requirement cannot be met by traditional phase conjugate mirrors based on photorefractive crystals whose gain is typically much less than one27,28.
Moreover, the significant challenge of undesired background illumination due to partial phase conjugation needs to be addressed. With complete time-reversal, the TRUE focusing technique can be conceptually represented as photons retracing their paths back to the location of the virtual source. However, this view disregards the wave-nature of light: complete time reversal requires full control over phase, amplitude and polarization of the entire scattered field over the full solid angle—which is fundamentally unfeasible (see Section I.1). As a result, even with perfectly aligned optics and noise-free recording of the scattered wavefront, the time-reversed focus is necessarily accompanied by a background29-31 which would obscure the fluorescence signal originating at the desired optical focus.
One or more embodiments of the present invention present a new strategy to overcome these challenges by combining digital phase conjugation32 with dynamic wavefront manipulation. The formation of an optical focus can be directly visualized, exciting fluorescence between layers of highly scattering tissue. This provides confirmation of the presence of the accompanying background, predicted by theory, that can be dynamically reproduced and subtracted. This digital background cancellation procedure, along with the high phase conjugate gain and resolution of one or more embodiments of the technique, enable the first demonstration of focused fluorescence imaging 2.5 millimeters deep inside biological tissue.